Nuclear cardiac myosin light chain 2 modulates NADPH oxidase 2 expression in myocardium: a novel function beyond muscle contraction

Abstract

Recent studies demonstrated that NADPH oxidase 2 (NOX2) expression in myocardium after ischemia-reperfusion (IR) is significantly upregulated. However, the underlying mechanisms remain unknown. This study aims to determine if nuclear cardiac myosin light chain 2 (MYL2), a well-known regulatory subunit of myosin, functions as a transcription factor to promote NOX2 expression following myocardial IR in a phosphorylation-dependent manner. We examined the phosphorylation status of nuclear MYL2 (p-MYL2) in a rat model of myocardial IR (left main coronary artery subjected to 1 h ligation and 3 h reperfusion) injury, which showed IR injury and upregulated NOX2 expression as expected, accompanied by elevated H₂O₂ and nuclear p-MYL2 levels; these effects were attenuated by inhibition of myosin light chain kinase (MLCK). Next, we explored the functional relationship of nuclear p-MYL2 with NOX2 expression in H9c2 cell model of hypoxia-reoxygenation (HR) injury. In agreement with our in vivo findings, HR treatment increased apoptosis, NOX2 expression, nuclear p-MYL2 and H₂O₂ levels, and the increases were ameliorated by inhibition of MLCK or knockdown of MYL2. Finally, molecular biology techniques including co-immunoprecipitation (Co-IP), chromatin immunoprecipitation (ChIP), DNA pull-down and luciferase reporter gene assay were utilized to decipher the molecular mechanisms. We found that nuclear p-MYL2 binds to the consensus sequence AGCTCC in NOX2 gene promoter, interacts with RNA polymerase II and transcription factor IIB to form a transcription preinitiation complex, and thus activates NOX2 gene transcription. Our results demonstrate that nuclear MYL2 plays an important role in IR injury by transcriptionally upregulating NOX2 expression to enhance oxidative stress in a phosphorylation-dependent manner.

Magnesium Lithospermate B Derived from Salvia miltiorrhiza Ameliorates Right Ventricle Remodeling in Pulmonary Hypertensive Rats via Inhibition of NOX/VPO1 Pathway

“…Oxidative stress is usually caused by the overproduction of reactive oxygen species (ROS) and NADPH oxidases (NOX), particularly NOX2 and NOX4, which are considered as the major source of ROS in cardiovascular system. Studies from other labs and ours have repeatedly demonstrated that NOX-derived ROS contributes greatly to myocardial/cerebral ischemic injury [8][9][10], endothelial dysfunction [11,12], and vascular remodeling [13,14]. There is growing evidence that NOX is activated in animals or humans with PAH, indicating that NOX-derived ROS may contribute to the development of PAH [15][16][17].…”

Role of ALK5/SMAD2/3 signaling in the regulation of NOX expression in cerebral ischemia/reperfusion injury

“…A previous study identified that NOX2 and NOX4 are the major NOXs responsible for brain tissue ROS production in a rat ischemic stroke model (6). Numerous factors are reported to be associated with the regulation of NOX expression, including the transcription factor, nuclear factor (NF)-κB and myosin light chain (7). However, the detailed mechanism by which NOX expression is regulated has not yet been fully elucidated.…”

Upregulation of NOX2 and NOX4 Mediated by TGF-β Signaling Pathway Exacerbates Cerebral Ischemia/Reperfusion Oxidative Stress Injury

“…However, our previous studies found that NOX2 and NOX4 were the major subtypes responsible for brain tissue ROS production in a rat model of ischemic stroke [8]. Presently, many factors have been reported to be involved in the regulation of NOX expression, including transcription factor NF-κB and myosin light chain [9]. However, the detailed mechanism underlying NOX expression regulation is not fully elucidated.…”